Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent d-lactate dehydrogenase from Lactobacillus delbrueckii toward utilization of NADPH. Through one round of selection, we obtained multiple enzyme variants with superior NADPH-dependent activities and protein expression levels; these mutants may serve as important tools in biomanufacturing d-lactate as a renewable polymer building block. Importantly, sequence analysis and computational protein modeling revealed that diverging evolutionary paths during the selection resulted in two distinct cofactor binding modes, which suggests that the high throughput of our selection system allowed deep searching of protein sequence space to discover diverse candidates en masse.

中文翻译：

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基于氧化还原平衡原理的NADPH依赖反应的高通量体内选择平台的开发。

进行厌氧发酵的细菌必须保持氧化还原平衡。基于该原理的体内代谢进化方案仅限于靶向NADH依赖性反应。在这里，我们基于大肠杆菌中经过工程改造的产生NADPH的糖酵解途径，为体内的NADPH消耗反应开发了一种简便，特异性和高通量的基于生长的选择平台。我们将选择系统用于德氏乳杆菌的NADH依赖的d-乳酸脱氢酶的定向进化，以利用NADPH。通过一轮选择，我们获得了具有优异的NADPH依赖性活性和蛋白质表达水平的多种酶变体。这些突变体可作为生物生产d-乳酸的重要工具，作为可再生聚合物的组成部分。重要的，